Yaker Linda, Tebani Abdellah, Lesueur Céline, Dias Chloé, Jung Vincent, Bekri Soumeya, Guerrera Ida Chiara, Kamel Saïd, Ausseil Jérôme, Boullier Agnès
MP3CV-UR7517, CURS-University of Picardie Jules Verne, Amiens, France.
INSERM U1245, CHU Rouen, Normandie University, UNIROUEN, Rouen, France.
Front Cell Dev Biol. 2022 Mar 9;10:823450. doi: 10.3389/fcell.2022.823450. eCollection 2022.
Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate among patients with diseases such as atherosclerosis and chronic kidney disease. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs). Recent studies have shown involvement of EVs in the inflammation and oxidative stress observed in VC. We aimed to decipher the role and mechanism of action of macrophage-derived EVs in the propagation of inflammation and oxidative stress on VSMCs during VC. The macrophage murine cell line RAW 264.7 treated with lipopolysaccharide (LPS-EK) was used as a cellular model for inflammatory and oxidative stress. EVs secreted by these macrophages were collected by ultracentrifugation and characterized by transmission electron microscopy, cryo-electron microscopy, nanoparticle tracking analysis, and the analysis of acetylcholinesterase activity, as well as that of CD9 and CD81 protein expression by western blotting. These EVs were added to a murine VSMC cell line (MOVAS-1) under calcifying conditions (4 mM Pi-7 or 14 days) and calcification assessed by the o-cresolphthalein calcium assay. EV protein content was analyzed in a proteomic study and EV cytokine content assessed using an MSD multiplex immunoassay. LPS-EK significantly decreased macrophage EV biogenesis. A 24-h treatment of VSMCs with these EVs induced both inflammatory and oxidative responses. LPS-EK-treated macrophage-derived EVs were enriched for pro-inflammatory cytokines and CAD, PAI-1, and Saa3 proteins, three molecules involved in inflammation, oxidative stress, and VC. Under calcifying conditions, these EVs significantly increase the calcification of VSMCs by increasing osteogenic markers and decreasing contractile marker expression. Our results show that EVs derived from LPS-EK-treated-macrophages are able to induce pro-inflammatory and pro-oxidative responses in surrounding cells, such as VSMCs, thus aggravating the VC process.
血管钙化（VC）是一种心血管并发症，在动脉粥样硬化和慢性肾病等疾病患者中死亡率很高。在VC过程中，血管平滑肌细胞（VSMC）发生成骨转化并分泌异质性的细胞外囊泡（EV）。最近的研究表明，EV参与了VC中观察到的炎症和氧化应激。我们旨在阐明巨噬细胞衍生的EV在VC期间对VSMC炎症和氧化应激传播中的作用及作用机制。用脂多糖（LPS-EK）处理的巨噬细胞小鼠细胞系RAW 264.7用作炎症和氧化应激的细胞模型。通过超速离心收集这些巨噬细胞分泌的EV，并通过透射电子显微镜、冷冻电子显微镜、纳米颗粒跟踪分析、乙酰胆碱酯酶活性分析以及蛋白质印迹法分析CD9和CD81蛋白表达来进行表征。将这些EV在钙化条件下（4 mM Pi - 7天或14天）添加到小鼠VSMC细胞系（MOVAS-1）中，并通过邻甲酚酞钙测定法评估钙化情况。在蛋白质组学研究中分析EV蛋白含量，并使用MSD多重免疫测定法评估EV细胞因子含量。LPS-EK显著降低巨噬细胞EV的生成。用这些EV对VSMC进行24小时处理可诱导炎症和氧化反应。LPS-EK处理的巨噬细胞衍生的EV富含促炎细胞因子以及CAD、PAI-1和Saa3蛋白，这三种分子参与炎症、氧化应激和VC。在钙化条件下，这些EV通过增加成骨标志物和降低收缩标志物表达显著增加VSMC的钙化。我们的结果表明，LPS-EK处理的巨噬细胞衍生的EV能够在周围细胞（如VSMC）中诱导促炎和促氧化反应，从而加重VC过程。
